Abstract
Regulatory monographs in Europe and the United States require drug products for parenteral administration to be “practically free” or “essentially free” of visible particles, respectively. Both terms have been used interchangeably and acknowledge the probabilistic nature of visual particle inspection. The probability of seeing a particle in a drug product container varies according to the size and nature of the particles as well as container and inspection conditions. Therefore, the term “without visible particles” can be highly misleading in the context of what is practically achievable. This may lead to differences in understanding between industry practitioners and regulatory agencies. Is this term intended to mean “zero particles”, or is there any intention to distinguish between particle type such as “zero extraneous visible particles” or “zero proteinaceous particles”? Furthermore, how can “zero” particles as a criterion for release testing be reconciled with “practically free from particles” as stated in the definition and a low, justified level of proteinaceous particles after production?
The purpose of this position paper is to review best practices in the industry in terms of visual inspection process and associated operator training, quality control sampling, testing, and setting acceptance criteria corresponding to “practically free of visible particles” and providing considerations when visible proteinaceous particles are deemed unavoidable. It also provides a brief overview of visible particle characterization and gives perspectives on patient safety. This position paper applies to biotechnology-derived drug products including monoclonal antibodies in late-phase development to licensed products.
LAY ABSTRACT: In the 2011 monoclonal antibody monograph revision, European Pharmacopoeia experts acknowledged that protein products may also contain proteinaceous particles at release or that protein particles may form during storage. Indeed, industry experience has demonstrated that therapeutic proteins such as monoclonal antibodies can exhibit a propensity for self-association leading to the formation of aggregates that range in size from nanometres (oligomers) to microns (subvisible and visible particles). As a result, the requirement for drug product appearance for monoclonal antibodies was changed from “without visible particles” to “without visible particles unless otherwise authorised or justified”. In our view, “practically free from particles” should be considered a suitable acceptance criterion for injectable biotechnology and small-molecule products, as long as appropriately defined. Furthermore, we argue that visual inspection is a suitable quality control release test and that “practically free from particles” is a suitable specification when adequately described.
- 100% inspection
- Lyophilized
- Quality control sampling
- QC sampling
- Particle identification
- proteinaceous particles
- visible particles
1. Problem Statement
The European Pharmacopoeia (EP) and United States Pharmacopeia (USP) monographs for parenteral preparations require drug products for parenteral administration to be “practically free” or “essentially free” of visible particles, respectively. Both terms have been used interchangeably. These definitions acknowledge the probabilistic nature of visual particle inspection. The EP monograph 2031, Monoclonal Antibodies for Human Use, as revised in 2011 set the following expectations and requirements for visible particles in monoclonal antibody drug products, as noted below in excerpts from different sections in the monograph:
Definition: “Examined under suitable conditions of visibility, they are practically free from particles.”
Production: “As part of the in-process control each container (vial, syringe or ampoule) is inspected after filling to eliminate containers that contain visible particles. During development of the product it must be demonstrated that either the process will not generate visible proteinaceous particles in the final lot or such particles are reduced to a low level as justified and authorised.”
Tests—Appearance: “They are without visible particles, unless otherwise justified and authorised.”
The question remains unresolved for a clear meaning of the term “without visible particles”; this may lead to differences in understanding between industry and agencies. Is this term intended to mean “zero particles”, or is there any intention to distinguish between particle type such as “zero extraneous visible particles” or “zero proteinaceous particles”? Furthermore, how can “zero” particles as a criterion for release testing be reconciled with “practically free from particles” as stated in the definition and with a low, justified level of particles after production? Is zero particles a consistent, realistic outcome?
PHARMEUROPA Vol. 23, No. 3, July 2011 provided explanatory notes and summary of changes to the monograph 2031 to be published in Supplement 7.3.
The merit of the 2011 monoclonal antibody monograph revision was that EP experts acknowledged that protein products may also contain proteinaceous particles at release or that protein particles may form during storage. Indeed, industry experience has demonstrated that therapeutic proteins such as monoclonal antibodies can exhibit a propensity for self-association, leading to the formation of aggregates that range in size from nanometres (oligomers) to microns (subvisible and visible particles). As a result, the requirement for drug product appearance for monoclonal antibodies was changed from “without visible particles” to “without visible particles unless otherwise authorised or justified”.
It was stated in the explanatory notes that “without visible particles” was intentionally kept to give clear guidance that the presence of visible particles is unwanted and the appropriate formulation studies should be performed during development to minimize visible particle formation; practically free could not be a pass/fail criteria in a test, and that visual inspection is not a quality control (QC) test, even though performed at the end of the production.
As a result of the probabilistic nature (1) of detecting particles by visual inspection method, “without visible particles”—meaning zero visible particles—is an unrealistic requirement for QC release/shelf life testing of any parenteral product and especially those of biotechnological origin. Even with significant formulation and container development, supported by long-term stability studies and stress stability studies (e.g., agitation), the probability of a visible particle being present cannot be completely eliminated. Interestingly, a requirement of without (zero) visible particles is not aligned with the requirement for small-molecule parenterals, which states “essentially/practically free” of visible particles.
In our view, “practically free from particles” should be considered a suitable acceptance criterion for injectable biotechnology and small-molecule products, as long as appropriately defined. Furthermore, we argue that visual inspection is a suitable QC release test and that “practically free from particles” is a suitable specification when adequately described.
The purpose of this position paper is to review best practices in the industry in terms of visual inspection process and associated operator training, QC sampling, testing and setting acceptance criteria corresponding to “practically free of visible particles”, and providing considerations when visible proteinaceous particles are deemed unavoidable. It also provides a brief overview of visible particle characterization, and perspectives on patient safety.
This position paper applies to biotechnology-derived drug products including monoclonal antibodies in late-phase development to licensed products.
2. Visual Inspection at End of Drug Product Manufacturing—100% Inspection Followed by Sampling Inspection
The presence of particles in parenteral finished drug products is dependent on the manufacturing process and manufacturing environment (design, qualification, validation, execution) as well as post-production handling, storage conditions, transportation, and handling by end users. This includes the choice and processing of primary packaging components, and also the design and stability of the formulation, particularly for biotechnology products. All products intended for parenteral administration must be visually inspected for various (critical, major, and minor) defects, including the presence of visible particles as required by the pharmacopoeia and current good manufacturing practice (cGMP).
The drug product manufacturing processes and associated controls are designed to yield, for example, single- or multiple-dose vials, syringes, cartridges, or ampoules containing solution or lyophilised solid of a formulated drug product that are, to the extent possible, free from visible particles.
As a last unit step (mandatory 100% inspection) during the post-filling operation, every unit of filled containers is visually inspected for critical, major, and minor defects including the presence of visible particles. For products with specification of “practically free from visible particles”, any filled unit found with a critical or major defect is rejected. Visible particles are classified as at least a major defect; refer to Section to 3.3 for exceptions.
Visual inspection is conducted according to USP chapters <1>, <790>, and European Pharmacopeia 2.9.20 using manual, semi-automated, or automated equipment. Of note, inspection conditions defined in EP/USP monographs are at least 2000–3750 lux, black and white backgrounds, and at least 5 s viewing against black and white backgrounds.
Detection of visible particles is a statistical process and, in routine practice, the detection limit depends on the individual operator, the inspection system, the inspection time, properties of the particle such as morphology and number, properties of the solution such as viscosity rheology, opalescence, and refractive index of the solutions, as well as other factors including the primary container (e.g., container size and shape, fill volume).
There is no single size cut-off for a particle being visible to the human eye. The detection limit for particles in a product is proportionate to the probability of detection such that the detectable size range varies from less than 50 μm, at near 1% probability of detection, to 200 μm, for >95% probability of detection under experimental conditions. As described, the probability of detecting a particle is influenced by several factors. For example, detection of 50 μm spherical polystyrene particles is at an approximately 1% probability of detection (2), whereas a 100 μm particle in 1 mL solution, in a 1 mL glass syringe, can be detected at about 70% detection probability. Size cut-off may be higher based on the nature of particles and other factors. Detection limits may be specified in company-specific procedures.
Also, companies may deviate from pharmacopoeial methods for manual visual inspection to improve the sensitivity or ergonomics of the process, such as the use of aids, for example, anti-glare glasses, observation times, or swirling procedures; variation in light intensity is already a flexibility provided in pharmacopoeia. These method operational parameters may have a pronounced effect on reject rates.
Slightly opaque or coloured container materials, for example, opaque plastic syringes, typically require use of a higher light intensity (e.g., 8000–10,000 lux) during 100% inspection and subsequent sampling testing to successfully detect visible particles. Highly coloured containers may require transfer to a clean, transparent container for visual inspection.
Visual inspection operators are trained (Appendix 1) using several runs of defect sets that may include various particle types, for example, product contact materials (stainless steel, aluminum, glass, rubber), particle size standards and fibres, hair, and so forth. The specific design, and also the stability, of these test kits need to be described in company-specific standard operating procedures (SOPs) and may be tailored to suit a specific process and product.
An operator must be able to detect a predefined proportion of defects in the training set (not exceeding a maximum amount of false positives) in order to be qualified (Appendix 1).
During routine 100% inspection, all units with detected defects are segregated, classified, and counted. All units with confirmed particle defects are discarded; refer to Section 3.3 for exceptions.
For products “practically free of visible particles”, any unit with an observed visible particle defect irrespective of its nature is discarded at 100% inspection, and this applies to liquid and lyophilisate. Products specified as “may contain proteinaceous particles”, if justified, can accept protein particle at 100% inspection with appropriate training and certification through use of product-specific training sets and guidance materials, for example, photographs/video, actual examples of the proteinaceous particles for direct comparison.
One hundred percent inspection may be manual, automated, or a combination. When automated inspection is used, ejected units can be manually inspected to verify the defect (e.g., presence of particulate material from other particle phenomenon such as micro-bubbles), and the detected defects are classified according to company procedures. Ejected units confirmed for the presence of a defect such as visible particles would be rejected; refer to Section 3.3 for exceptions.
Biopharmaceutical manufacturers are expected to have assessed the capability of their commercial fill/finish process for each product (and strength) at each manufacturing facility. This assessment often includes 100% inspection results, and sponsors often use this capability to set action limits, or a maximum allowable reject rate, for the visible particle defect rate in commercial products. If the maximum defect rate is exceeded then an investigation is initiated. For clinical products, a defect rate criterion may not be viable given the limited manufacturing history. For manufacturing trending, bracketing and matrixing approaches can be used, for example, performing trending only for the highest and lowest dose strength (not every single dose strength) or, for example, clustering of comparable product families.
The 100% inspection is unable to assure detection of all visible particles due to the direct relationship between the probability of visual detection and particle size (as well as other parameters). Therefore additional assurance is provided by a second inspection based on statistical sampling—for example, acceptable quality limit (AQL), sampling based on predefined sampling plans, such as ANSI Z1.4, ISO 2859-1 or better or equivalent. This second inspection is performed by different inspectors to those performing 100% inspection.
The additional visual inspection test based on a sampling plan is most commonly referred to as AQL (acceptable quality limit) testing. It might also be referred to as an acceptance sampling plan (ASP) in some companies.
The AQL chosen for use with sampling plans can be based on a survey of current inspection practices conducted by the Parenteral Drug Association (PDA) (3). This survey of both European and US-based manufacturers established that the industry median maximum AQL for the major (Level 2) defect category is 0.65%.
AQL maximum of 0.65% has been adopted in USP <790> and is typically used as the criterion for the AQL test, unless otherwise justified (4).
Circumstances dictate when switching to a higher level of testing is required, which could mean an increased sample size and lower maximum AQL for a tightened sampling plan. A tighter sampling plan may be triggered by detection of an atypical particle type, or increased particle detection frequency, suggesting procedural issues that may require corrective and preventive actions (CAPAs) and may reflect a system failure that presents greater risk to patients.
ANSI Z1.4 and ISO 2859-1 use AQL tables to determine the appropriate sample size; however, other methods may also be used that result in an acceptable AQL for visual inspection. Methods also exist that have reduced dependence on batch size, for example, use of operating characteristics curves as described by Knapp and Budd in 2005 or Taylor in 1992 (5, 6).
As an illustrative example of AQL testing: Applying a Level II ANSI/ASQ Z1.4 plan for batch sizes of 10,001 to 35,000, the batch is considered to be “practically free of particles” if no more than five (5) units are observed to contain one or more visible particles in a sample of 315 units examined under the conditions described above. Detection of six (6) units with visible particle(s) would fail the test. Visual inspection Level II testing may be considered suitable for detection of visible particles when product development did not reveal concerns of visible particle generation. Additional investigation of particles detected during AQL testing would not be required unless they are unexpected, atypical particles that suggest system failure (e.g., insect parts), in which case it is recommended to trigger further investigation.
As an alternative to directly using the AQL tables provided in ANSI/ASQ Z1.4, operating characteristic (OC) curves may be generated for the proposed sampling plans with parameters adjusted to meet the desired AQL and unacceptable quality limit (UQL). The OC curve then determines the sample size and accept/reject numbers. The impact of lot size on a sampling plan is minimised by fixing both AQL and UQL and ensuring that the sample size (n) is less than 0.1 × N, where N is the lot size.
Batches that fail AQL criteria may be considered for 100% re-inspection dependent upon the outcome of an investigation and on specific company procedures.
Should 100% inspection be repeated, another AQL-based inspection would need to be passed and may use tighter AQL criteria such as increased sample size. The tiers of retesting should be limited and described in company procedures (usually not more than two cycles of re-inspection). Ultimately, repeated test failure may result in batch rejection.
When 100% inspection action limits show special cause variation (6, 7) in the process, additional AQL samples may be required to provide additional assurance of quality.
When the batch size is small (e.g., less than 501 units) then reduced, special sampling plans may need to be considered and justified by the sponsor. The Special Sampling Plans described in ANSI Z1.4/ISO 2859-1 may also be considered.
For product with a history for formation of proteinaceous particles, units containing visible proteinaceous particles may be accepted during the inspection provided that these expected protein (product-related) particles can be differentiated from foreign matter by a qualified procedure. This could include specific training and use of guidance materials for visual identification of known, expected particle types including examples of product particles from that manufacturing line, in connection with specific analytical testing.
The visual inspection process is depicted in Figure 1 for a liquid product (liquid-filled units with integral container closure) to be classified as practically free from visible particles.
Schematics of typical visual inspection process—liquid-filled units.
Two key concepts—that is, presence of particles qualified as “occasional random occurrence (first concept) or representative of a system failure (second concept)—are introduced in this flow chart and further elaborated in Appendix 2:
A batch may be passed with occasional, random occurrence of particles if these are within the AQL acceptance criterion.
Any critical visible defect issues with regards to nature of particles and representing a system failure (insect wing, rust, paint, hairs, etc.) will lead to an investigation/re-inspection.
If the investigation indicates the potential for sterility breach during aseptic processing, this type of system failure may lead to batch rejection.
To summarize this section, 100% inspection (whether done manually, semi-automated, or automated) does not result in 100% of units without any visible particles. Although zero defects is the desired goal and should drive continuous process improvement, it is not a workable acceptance criterion for visible particles because of current capabilities in the industry for packaging components, processing, and facility. In addition, protein particles may be an inherent quality attribute for protein biologic products particularly at high protein concentrations, despite efforts to develop an optimized formulation and container system.
Even when using state-of-art technology and approaches, an absolute statement that all units will be “free from visible particles” cannot be made. It is for this reason that the USP and EP in their general monographs on sterile injectable products use the phrases “essentially” or “practically” free of visible particles.
The “practically free from visible particles” requirement is intended to specify a particle-free product while considering the limitations of the visible particle inspection process, thus acknowledging that all drug product units cannot be “without” particles. Based on the above concepts, our suggestion is to improve clarity and wording of the monoclonal antibody monograph in the EP by reflecting this concept and, for example, adopting language to reflect that during QC, “drug product units are practically free of visible particles, unless otherwise justified and authorised”.
3. QC Sample Testing
This section discusses best practice approaches for the QC assessment of visible particles for product batch release and stability testing of parenterals products containing biotechnology-derived active pharmaceutical ingredients, including monoclonal antibodies. This includes liquid products, coloured or opaque containers, and drug/device combination products such as prefilled syringes (PFSs), pens, autoinjectors, and delivery pumps. Sampling plan recommendations are provided for biologics when “practically free from particles” is specified. These principles may be applied to parenteral drug products in general. Considerations are also provided for the control of products that may contain proteinaceous product particles. Lyophilised products are discussed in Section 4.
3.1. Products “Practically Free From Particles”
When the product is specified as “practically free from particles”, batch testing may be achieved using different strategies depending on the manufacturing in-process and QC batch release control procedures in place. Therefore, different approaches to QC control of visible particles are considered equally applicable.
3.1.1. Post-100% Inspection, AQL Testing May Replace End-Product Testing:
For clear, liquid dosage forms in clear containers using non-destructive visual inspection, end-product release testing may take the acceptance sampling plan (AQL) result as sufficient and appropriate to confirm the specification of “practically free from particles”. For products that require destructive testing for visual inspection, refer to Sections 3.4 and 4. The approach of moving the specified control of visible particles upstream to the manufacturing in-process AQL test requires that the AQL inspectors are trained for the detection of visible particles, to the same level as would QC end-product release testing inspectors (Appendix 1) for particle detection endpoints. Depending on the timeframe between manufacture and the AQL testing, use of post-100% inspection AQL testing to support compliance of a batch to its visible particle specification may also be considered as a real-time release testing (RTRT) alternative to end-product QC release testing. Conformance of RTRT to cGMP can be confirmed at the manufacturing site inspection and should permit relief from EU import testing when manufacture is performed in a non–EU member state without a Mutual Recognition Agreement in place.
3.1.2. Batch Release End-Product QC Testing:
When a drug product has successfully passed 100% inspection and AQL testing during manufacture, a product is typically released on the basis of end-product testing in a QC environment.
As described in Section 3.1.1, the AQL result alone may be justified as suitable for batch release with no further end-product release testing for visible particles. Alternatively, if a successful AQL result is not being used for batch release, then another confirmatory visual inspection may be performed using a predefined sample size, for the purpose of QC release (Section 3.1.3).
3.1.3. Sampling Sizes:
A survey among the biopharmaceutical manufacturers contributing to the elaboration of this position paper suggested that in many cases, 10 to 20 samples have been used to inspect for QC batch release when end-product testing was required.
3.1.4. Reuse of Visually Inspected Units:
Samples used for visual inspection may be reused for other QC tests (e.g., purity, protein content) if desired and justified.
Re-use applies to both non-destructive or destructive testing when the container is opened and content manipulated by, for example, reconstitution or transfer to a clean container (see also Section 4. Control of Visible Particles in Lyophilized Products). However, stability of these samples during visual inspection needs to be assessed, and it needs to be ensured that conditions the sample is exposed to during analysis, for example, temperature cycling and sample handling and visual testing assessment, is not affecting subsequent analyses and results.
3.2. Use of In-Line Filters during or Prior to Administration
In general, the presence of visible particles may be mitigated, for example, by use of a filter during intravenous or even subcutaneous administration. This is also acknowledged in the respective EP and USP monographs. However, the compatibility of a product solution and effectiveness of an in-line filter to reduce particles should be qualified by the sponsor. Furthermore, the use of in-line filters cannot be used to justify accepting units with any type of visible particles during 100% manufacturing inspection and AQL testing. In-line filters may be used with products that are expected to form protein particle after adequate justification (see Section 3.3 for a discussion on protein particles).
The end-product release specifications would, in these cases, possibly change from “practically free of visible particles” to an acceptance criterion reflecting the presence of (protein) particles once sufficient manufacturing and stability knowledge is gained. EU and US product information would then be consistent with the proposed acceptance criteria (see Section 6).
3.3. Products that may contain Proteinaceous Particles
Biologics, including monoclonal antibodies, can have an inherent molecular property to self-associate, or aggregate and form proteinaceous particles despite formulation, manufacturing process, and container closure development to minimise visible protein particles. This propensity is a basic thermodynamic property of the molecule that cannot be totally overcome. Such particles may form over time, often exist in equilibrium, and may or may not be reversible as a result of non-covalent interactions. Many biologic therapeutic agents require several milligrams per killogram of dose for efficacy, resulting in tens to hundreds of milligrams protein to be administered. For a subcutaneous injection this requires formulation of a high-protein concentration of product with appropriate formulation to achieve acceptable physical properties such as viscosity, and to provide a stable product with minimal aggregate and particulate content (high-molecular-mass species, sub-visible particles, and visible particles). Increasing protein concentration can promote molecular interactions and hence protein particle formation.
The Notes to the EP monograph for monoclonal antibodies for human use (EP 2031) recognises that final product “may contain proteinaceous visible particles that are intrinsic to the product”. If the sponsor can suitably justify that formulation development has been sufficiently performed to “minimise the presence of such visible proteinaceous particles”, then rare occurrence of formation of such particles may be considered an acceptable quality attribute after risk assessment concluding no impact to safety and efficacy.
If suitably justified (e.g., by risk assessment that includes evaluation of possible patient risks, such as immunogenicity, embolism, and other adverse effects), well characterised proteinaceous particles at a defined level and frequency may be considered acceptable in the drug product, even without use of an in-line filter (Section 5 and Appendix 2).
There can be various approaches for 100% inspection, AQL testing, and end-product batch release. For example, the level and frequency of visible proteinaceous particles is compared to the specification and documentation that describes the expected or allowed characteristics of particles for that product. This procedure would allow the presence of units with protein particles that are similar to those expected and within a defined frequency. Any unit with a non-protein particle would continue to be discarded. Should the defined frequency of particles be exceeded, then the non-conformance should trigger investigation leading to potential batch rejection. Specific training, using product-specific test panels, including actual product manufacturing line samples, and photographs/videos and so forth, may be used to qualify and validate an appropriate procedure to ensure that certified inspectors can differentiate proteinaceous from non-protein particles based on a different morphology and behaviour, in connection with adequate analytical technology.
End-product QC release testing of a protein with expected inherent protein particles may use the AQL testing results after successful 100% inspection without requiring an additional, confirmatory QC test.
When a product has a specification that allows for the presence of proteinaceous particles, a means of quantification to detect changes that may occur becomes more challenging, and the instructions should be outlined in company procedures.
To support the overall control strategy for visible particles and justification for the presence of low-level protein particles at QC release, it is recommended that the product's development and manufacturing history is adequately documented, including the potential occurrence of visible particles during manufacturing, stability, and transportation-related handling as well as any clinical findings and adverse events. This history may derive from separate documents according to company practice or constitute the product pharmaceutical development report.
The particle history would vary according to the particle types detected. In documenting the particle history, it may be useful to include information on particle detection trends, for example, frequency across a lot, and number of particles per container if possible; description and characterization (e.g., buoyancy, composition) data on detected particles and records of actions taken; risk management plan for the presence of any “expected” (previously detected for the product) and “unexpected” (novel, not previously detected) visible particles; and support and justification for the visible particle specification. It is acknowledged that particle history might be documented differently depending on company-specific pharmaceutical and quality systems requirements.
3.4. Considerations for Opaque or Coloured Products or Primary Packaging Material and Products with Restricted View
Products that are slightly opaque and products in slightly coloured or opaque primary containers may use higher light intensity for visual inspection for visible particles as permitted in EP 2.9.20 and USP <790> (e.g., 8000–10,000 lux). For AQL testing, products in coloured or opaque primary containers may be transferred to clean, transparent containers. Also, for products that are highly opaque or coloured, dilution may be required. Extra care is required, such as use of particle-free water and aseptic sample handling in a controlled environment, when transferring or diluting the product to avoid introduction of foreign, extraneous particles. Similar sampling considerations as presented above for liquid drug product should apply.
It may not be readily feasible to perform visual inspection of PFSs once assembled into a pen, autoinjector, infusion pump, or patch device if the prefilled primary container of drug cannot be easily removed, for example, the device assembly components lock the PFS inside. In this case, it is sufficient to inspect the PFS, cartridge, or other primary container type prior to assembly to demonstrate that the assembled product is “essentially free from visible particles” provided that the assembly process steps are shown not to introduce particles into the drug product container closure system.
The inspection window in a device might be inadequate for visual inspection and particle detection and therefore a method qualification for visual inspection may not be possible. Destructive disassembly is often not recommended because the procedure risks affecting the container closure integrity and creating other cosmetic defects such as scratching, but it may be performed if the procedure is suitably qualified. If the product may be easily disassembled for inspection and analysis, without a possibility to affect container closure integrity and the procedure is scalable, then the container should be inspected at AQL testing, as described for a typical liquid product. Disassembly should not be required at 100% inspection. Similar procedures are recommended for other assembled drug/device combination products.
3.5. Visible Particle Testing on Stability
Ideally, formulation, product, and process development studies should result in “practically free from particles” product that remains consistent with this acceptance criterion throughout the claimed shelf-life.
However, although undesired, particles may form over time with slow kinetics, for example, proteinaceous particles or particles resulting from interaction with primary packaging (8⇓⇓–11) and/or excipients and due to shaking during transport. Also, the appearance of protein particles is typically not uniform across all containers in a batch as a function of time, and each container with protein particles will look slightly different from another. Additionally, the probabilistic nature of visible particle detection means that it is not possible to entirely discount the detection of pre-existing particles in stability testing when new units of product are being inspected at each time point of the stability protocol.
The detection of pre-existing particles in stability should be avoided because stability testing should focus on changes in the product over time and not discovery of particles that would have been present at time zero (an exception is a product with expected, inherent proteinaceous matter at release). Therefore, a pre-screen of stability samples may be performed to eliminate units with unexpected visible particles at time zero. If a product is justified to have proteinaceous particles at release, then that would constitute “expected” particles and be permitted in stability according to the stability specification.
In addition to the tendency of protein product to aggregate, other time-induced changes can occur that relate to the formulation and container closure materials of construction. One such phenomenon is the formation of visible glass lamellae resembling flake-like particles. When detected during product development, these visible particles should be avoided to the extent possible by formulation, process, and product development, including potentially considering changing the primary packaging washing/sterilization process, formulation and/or container, as these particles would indicate a system failure.
The QC release visual inspection result can be used for the time zero stability result. When the AQL result used for release is not suitable for stability testing, then additional units are required for the time zero visual inspection. The number of units tested at time zero and additional time points may depend on the properties of the finished product and should be justified in company procedures. Sampling sizes for QC stability testing differ across companies.
A survey among the biopharmaceutical manufacturers contributing to the elaboration of this position paper suggested that in many cases, 10 to 20 samples have been inspected during QC stability testing at each time point for both liquids and lyophilisates.
A product with no history of particle formation may justify stability sampling with a lower number of units than a product with a known tendency to form proteinaceous particles.
When the visual inspection test is not destructive, the same samples may also be used for subsequent stability time points tested. This must be supported by studies that assure product would not be at increased risk of particle formation over time as a result of sample handling stress during repeated inspection and that samples can be clearly traced to avoid mix-up of samples over the course of the stability programme. Defective samples may be replaced to maintain the sample size. Alternatively, separate units can be inspected for each time point, taking into consideration that unexpected, extraneous, visible particles may be found, as 100% and AQL inspection operations are probabilistic and single units with rare and randomly occurring particles may occur.
Stability samples used for visual inspection may be reused for other batch control tests or re-inspected at further time points, if it is demonstrated that the visual inspection does not affect other QC test parameters.
Semi-quantitative methods such as visual comparison against barium sulphate precipitate (12) or other standards and instrument-based methods are in development to evaluate the levels of inherent particles in a product. If these can be validated (13), such methods could possibly be introduced into release/stability product testing.
Given sufficient product understanding and history to assure stability over the proposed shelf-life, it may be justifiable to reduce frequency of testing for visible particles for annual batches placed into the product stability protocol.
4. Control of Visible Particles in Lyophilized Products
Lyophilised products present unique challenges for the control of visible particles because the cake will obscure detection of most visible particles. Therefore, 100% inspection, though still required and of value for general container and lyophilised cake appearance, has limited capability for detection of particles. Therefore, reconstitution of the product is to date recommended during QC for inspection for visible particles. Because the reconstitution is destructive to the sample, it cannot be performed for 100% inspection, nor is it feasible to use AQL sampling plans to the extent possible for a liquid product. A different approach is required for visible particle control for lyophilised product to a liquid product. At minimum a predetermined sample size of lyophilisate in stoppered, capped vials should be reconstituted, during end-product QC testing, as justified by the sponser. The sampling plan may be based on the current industry standard of 10 to 20 units, which is supported by the history of marketed lyophilised products, or by using ANSI Z1.4 (ISO2859-1) Special Sampling Plans. Equivalent or better sampling plans may be used.
Note that post-lyophilisation sample handling, for example, reconstitution, can itself result in false-positive detection of particles. The risk of introducing particles of any source during reconstitution is not pertinent to the control of particle during manufacture, product batch release, or stability testing and should be a concern of product handling and investigated as part of method development, container closure qualification, and compatibility with the product. Thus, reconstitution and handling in QC should take into consideration the instructions for use also provided for the reconstitution by end users (e.g., handling by health care providers).
Visual inspection for lyophilisates in a stability programme again requires reconstitution of a predetermined sample size at each time point, as described in Section 3.5.
Samples of reconstituted lyophilisate, after visual inspection, can be also used for other QC tests, with the same considerations applying as for liquid products.
5. Particle Identification and Characterization
The presence of visible particle(s) in a drug product container may drive an investigation that often includes further characterization to determine particle attributes, such as size, morphology, and composition, in an effort to find root cause. An investigation would be triggered when, for example, atypical particles are detected, unexpected growth of inherent protein particles over time is observed, or if acceptance criteria of units with visible particles are exceeded. Particle characterization and identification typically involve research methods that are qualitative in nature, and these methods are not recommended as routine tests, such as assays for batch release.
The origin of the visible particles (14) can include environmental, process, packaging components, the protein drug itself, and any combinations of these. In the cases of atypical findings, the knowledge of the particle origin may support the risk management and control strategy and drive CAPA.
Non-conformance investigations as wejll as advanced particle characterization as part of product and process development may require the use of a combination of various analytical techniques. A multi-pronged approach may be necessary because a single analytical technique may not be able to reveal the key attributes of a particle. A first step is to determine the size, number, and aspect of the particle(s) in the unopened primary container, to the extent possible. Focused visual inspection under enhanced visualization conditions (such as polarized light and/or magnification, stereo- or inverted-light microscopy, advanced image analysis) can be used as additional analytical tools for non-destructive analyses. Particle attributes from nondestructive analyses such as size, number, shape (e.g., fiber, sphere), color, type (e.g., metallic, glass) and settling behavior may support particle identification.
However in some rare instances, further analytical characterization may be necessary to gain more reliable information on composition and origin of the particles. These tests are destructive in nature (i.e., requiring opening of the container) and may often involve isolation of the particle(s) for further characterization. Therefore, careful sample handling is very important to maintain integrity of the particle(s) planned for identification and ensuring no introduction of environmental particles during handling and analysis. In some cases, the dosage form may contain a single particle or a very few particles. Furthermore, it is quite common that only a single container (with atypical particle) is available for detailed characterization. Required sample volume for such extended characterization tests is also an important consideration, especially in early stages of development. Therefore, a predetermined strategy should be considered to ensure adequate data can be collected using limited number of containers and/or limited volume of sample.
For example, the tests that are non-destructive in nature (not requiring container opening) should be conducted first (see flow chart in Figure 2). These include visual inspection as well as use of enhanced visualization conditions such as magnification, lighting, contrast, and optical microscopy. Subsequent tests for further characterization will require opening of the container. Physico-chemical characterization of the nature and composition of particles can be accomplished following the filtration of the particles on appropriate membrane or other sampling techniques. The sample handling step is critical and needs to be carefully controlled because it may alter the particle by, for example, oxidation or breakage or dissolution of fragile particles, and result in additional particle ingress (false positives) or in a loss of the particle of interest. Tests with the isolated particle(s) may include optical microscopy for detailed morphological assessment, Fourier transform infrared (FTIR) or Raman microscopy for compositional analysis, and other techniques such as Time-Of-Flight Secondary Ion Mass Spectroscopy (TOF-SIMS) that may be applicable for certain cases. In this stage of the analyses, isolated particles are not subjected to significant treatments other than isolating/filtering. If further characterization is needed and sufficient material is available, then as the last step in the characterisation process, isolated particle(s) can be biochemically treated to enable tests such as Matrix Assisted Laser Desorption Ionization (MALDI), sodium dodecyl sulfate capillary electrophoresis (CE-SDS), Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), peptide mapping, and the like.
Flow chart for usage of containers for particle characterization.
Visual inspection as well as enhanced visualization tests provide a good clue of the type of particle based on shape/morphology. For example, protein particles are often fluffy, amorphous, and translucent in nature, and may not reflect well under polarized light inspection conditions. Protein particles also can be fibrillous (fiber-like). On the other hand, glass and metal particles are often shiny and/or crystalline in nature.
Extended characterization tests for composition and origin of particles include FTIR microscopy and Raman microscopy. FTIR/Raman microscopic tests are suitable for measuring morphology of visible particles as well as compositional analysis through spectral fingerprinting. Several types of particle compositions including proteinaceous, inorganic, and organic can be studied by FTIR/Raman microscopy. These tests may also be suitable to study subvisible particles in low-micron size range (approximately >20 μm). In Raman microscopy assay, it needs to be considered that laser-induced damage of the sample can likely occur. Light microscopy tests using polarized light or fluorescence microscopy (with and without staining or labeling of particles) may provide additional insight into the nature and characteristics of the particles.
Additional supporting tests may include elemental analyses such as by scanning electron microscopy (SEM) coupled with energy-dispersive (EDX) or wavelength-dispersive X-ray detectors. However, SEM-EDX elemental data may not be confirmatory because particles of different origins may have similar elemental composition. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) imaging have also been described to provide supporting information about the particle morphology and fine structure at the molecular level (e.g., by cryo- or negative staining TEM). Again, careful sample handling and understanding method capabilities are important considerations for these tests.
The analysis of the size distribution (size and number of particles at each size) is particularly important for proteinaceous particles, which cover a wide range from visible precipitates to soluble sub-micron aggregates (15⇓–17). During product development, it is important to study a wide size range of aggregates/particles to understand a possible mechanistic link of small aggregates and subvisible/submicron particles to the formation of visible particles. Light-obscuration, flow microscopy, electrical sensing zone, and flow cytometry techniques are suitable to detect and count small amounts of particles in a size range from approximately 1 to 100 μm. These analytical techniques provide comparable information of size distribution trends, but particle counts may vary by orders of magnitude. Techniques to measure particles in the sub-micron size range include several emerging techniques such as field flow fractionation, nanoparticle tracking analysis, and resonant mass measurements. This is an active area of research, and robustness of these techniques are yet to be established.
In summary, establishing the nature or origin of visible particles is a complex process that requires thorough analytical due diligence. A toolset of various orthogonal analytical techniques provides the best opportunity to characterize particles, particularly the proteinaceous particles. The identification of visible particles should be considered for individual investigations and not for routine or QC testing.
6. Patient Safety
Particles remain a critical quality attribute, as “essentially/practically free of visible particles” is a requirement for parenteral dosage forms (unless, e.g., used with a filter during administration or when particles are a designed part of the formulation, e.g., insulin protein crystals). Various clinical empirical observations have been connected to particles in parenteral products, although there are no systematic studies that have correlated specific types and levels of particles to specific adverse effects. Literature in the field is mostly related to drug abuse or anecdotal evidence. A connection of (sub-visible or visible) proteinaceous particles and immunogenicity has been hypothesized, although there is no direct clinical evidence for this hypothesis. Clinical relevance of particles has been reviewed in recent literature (18, 19).
In general, small number of particles in parenteral products likely do not lead to any adverse impact on safety. This is corroborated by a review of the Product Information for monoclonal antibody products approved through the centralized procedure in the European Union and through Biological License Application in the Unites States of America (see Table I): In the table above, particle presence is referred to as “may”, emphasizing the unpredictable and probabilistic nature of these particles.
Examples of EU/US Product Information for Biotechnology Products Potentially Containing Protein Particles
Where unavoidable, an acceptance limit for proteinaceous visible particles should be based on exposure and adverse event data from non-clinical and/or clinical studies, assuming the product is not intended to be administered to the patient through an in-line filter as prescribed in the Product Information.
7. Conclusions
Visible particle assessments remain a challenge in the development of parenteral products, especially of biotechnology products. The biopharmaceutical industry is striving towards improving the quality and cleanliness of products, including addressing the upstream sources of particles. Although the nature of visual inspection is probabilistic and the presence of trace levels of particles in parenteral products cannot be fully excluded, a holistic approach to minimizing the presence of visible particles in parenteral protein drug products as for other injectable drug products is proposed. However a requirement of zero (or without) visible particles is overly stringent and practically not attainable. Particle controls are and should be one of the main formulation and process design criteria applied by the biopharmaceutical industry. This continuous improvement objective being acknowledged across the industry, the current industry position reflected and justified at length in this position paper is to consider “practically free from visible particles unless otherwise justified and authorised” as a standard requirement for QC release of biotechnology-derived drug products including monoclonal antibodies subject to compliance with the European Pharmacopoeia.
Conflict of Interest Declaration
The authors declare that they have no competing interests.
Acknowledgments
The paper was written in collaboration with other experts from European biopharmaceutical enterprises (EBE) Visible Particle topic group and from the EBE Biological Manufacturing Group member companies that contributed and supported the preparation of this document: Atanas Koulov, Analytical Development & Quality Control, Pharma Technical Development Biologics EU, Roche; Sharon Adderley, Manufacturing Science and Technology, Analytical Science, Pfizer; and Rober W. Kozak, Regulatory Affairs, Bayer Healtcare LLC.
Appendix 1: Inspector Certification
The visual appearance test is subjective and the result is probabilistic since the instrument is the analyst's eye. Effective inspector selection, training, and monitoring are therefore necessary for a reliable and consistent visual inspection program. The training for inspectors performing manual visual inspection of unlabelled drug product containers requires certification, which would include assessment of visual acuity and technical expertise that includes the ability to detect particles in test panels.
The same principle of training and monitoring should be provided for all personnel performing visual inspection for visible particles during manufacture as part of 100% manual inspection, AQL testing, QC release and stability testing, QA reserves/retention, and product complaints.
Inspector Selection and Qualification
Selection Criteria for Visual Inspector
Each trainee must have an eye exam completed, for example, including vision acuity test and color blindness test.
Trainees should pass eye examination before being qualified for visual inspection training. This documentation should be archived for future reference.
Training Process for Inspector
Introduction to general visual inspection method.
Introduction to visual inspection station with black and white background, according to PhEur 2.9.20.
If a defect library is available, a demonstration of typical defects should be performed. Defect set criteria and its stability, should be captured according to predefined SOP.
Demonstration of visual inspection method by trainer.
Demonstration of sample handling and swirling method, according to predefined SOP.
Introduction to defect categories and associated AQL levels as per SOP.
Proficiency runs using appropriate test panels (qualification defect test sets) for the process and product, as many as required.
Training sets (20) should include a range of defect types, including particles that would be relevant to defects possibly found during the manufacturing process. These test panels should use containers both with and without defects. It is recommended to include representative pictures or videos (with and without magnification) of the particle types as part of training material.
Training sets should be maintained using defined criteria. All sets should have expiration or retest date and may be reassessed, per SOP, for continued use. Note: Qualification conditions need to mimic the actual inspection conditions, for example, using the same rate of inspection for units and using the same equipment.
Maintenance and Monitoring of Visual Inspector
Regular, for example, annual, procedure review and assessment; compliance to SOP and ability to detect defects.
Eye exams must be performed regularly, for example, annually.
Inspector must be re-qualified at regular intervals, for example, annually, to be considered a qualified visual inspector.
Appendix 2: Risk Assessment of Visible Particles
Generally risk assessments evaluate the likelihood (occurrence) an event will happen (e.g., visible particle), the impact (severity) of the event on product safety and efficacy, and the ability to detect either the event or the impact.
The risk assessment should have input from all relevant disciplines—for example, product quality, CMC product development (process, formulation, analytical), regulatory, manufacturing, clinical, clinical pharmacology, drug safety, drug metabolism (pharmacokinetic/pharmacodynamics/immunogenicity), and pharmacovigilance.
The origin of the visible particle(s) may be environmental, related to the process, packaging components, drug product formulation or proteinaceous makeup and/or likely mixtures thereof. Categorization of particles into inherent, intrinsic, and extrinsic particles is defined in USP <790> monograph.
As mentioned in Section 5, in cases of atypical findings indicative of system failure, the knowledge of the particle origin/source may support the risk management and control strategy and drives CAPA. System failure is defined as opposite to “randomly and rarely occurring”. For example, the formation of glass lamellae resembling flake-like particles over time would signal a system failure (Section 3.6). Insect parts that might be detected during AQL testing at end of manufacturing would also be considered as particles reflective of a system failure. By contrast, as discussed in Sections 2 and 3, rare occurrence of particles or presence of proteinaceous particles is a concept that defines what might be considered as expected or normal based on the product history.
Based on batch history, and trend analysis of complaints, the likelihood of occurrence of proteinaceous particles can be determined as, for example, extremely rare, remote, occasional, likely, probable, almost certain, or even unknown, allowing differentiation of products that would fulfill requirements for being categorized as “product practically free from particles” from products that may contain protein particles.
For products “practically free from particles”, some isolated (occasional random) occurrence of visible particles (including protein particles) may occur, and be identified during QC testing at release or during stability studies or in complaints from patients/health care professionals and would trigger an investigation to identify the root cause, characterize the particles in order to assess possible safety and efficacy impact to patients taking into consideration the route of administration of the product, patient status, and so forth.
From a medical risk perspective, extraneous particles introduced by intravenous or intravitreal route has been a greater concern compared to intramuscular or subcutaneous routes. Proteinaceous particles may be a greater concern for subcuntanous administration (21) if a link of particles and immunogenicity is established. Particles injected into closed spaces (e.g., intrathecal, intraarticular) may not be eliminated or drained and also represent a higher potential for harm. Consequently the nature, size, and capability of the particles to dissolve or disaggregate might have to be assessed.
For particle characterization including protein particles, the following parameters may be considered:
Potential dissolution of the particles by swirling and exposure to room or even physiologic temperature.
Solubility, buoyancy properties of particles.
Characterization (size, number, volume, morphology) (see Section 5).
Quantity of particle (weight).
In the patient safety/efficacy assessment, the following parameters may be considered:
Use of in-line filter for administration to patients.
Adverse events: immunogenicity and impact of potential Anti-drug Antibodies (ADA), thromboembolic event (less of a risk of capillary occlusion with protein particle as they are likely flexible/deformable), intraocular inflammation, and so forth.
Daily intake—acute versus chronic therapy and exposure duration.
Population treated, patient general state, patient immune status.
Potential induction of pathological change.
Amount, size distribution of particles per individual container.
Dissolution and type of particles.
Possibility to migrate from the injection site to bloodstream.
Loss of potency.
Alteration of the pharmacodynamic effect.
Biodistribution.
Amino acid sequence similarity to endogenous proteins, permitting antibody cross-reactivity.
Ultimately the overall risk assessment can take into considerations the relevant parameters mentioned above to define a risk level associated with the particle(s) and support the decision for product disposition, and for preventive or corrective strategies. The overall risk level could be categorized as high, moderate, low, very low, or negligible.
- © PDA, Inc. 2016
References
Jump to section
- Article
- Abstract
- 1. Problem Statement
- 2. Visual Inspection at End of Drug Product Manufacturing—100% Inspection Followed by Sampling Inspection
- 3. QC Sample Testing
- 4. Control of Visible Particles in Lyophilized Products
- 5. Particle Identification and Characterization
- 6. Patient Safety
- 7. Conclusions
- Conflict of Interest Declaration
- Acknowledgments
- Appendix 1: Inspector Certification
- Appendix 2: Risk Assessment of Visible Particles
- References
- Figures & Data
- References
- Info & Metrics
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